There is a fundamental gap in understanding how PED/PEA-15 (phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocyte, 15 kDa), a small, non-catalytic protein, can regulate a number of distinct fundamental cellular processes, including apoptosis, extracellular regulated kinase (ERK) pathway, and glucose metabolism. This gap prevents the comprehension of the roles of PED/PEA-15 in tumor cell survival, proliferation and migration, resistant to chemotherapy, as well as the development of diabetes. The long-term goal is to elucidate the mechanisms of PED/PEA-15 in modulating remote biological pathways. The overall objective is to identify the functional roles of the charge triad, D-RxDL, a characteristic feature on almost all death effector domains (DEDs), of PED/PEA-15 in binding to ERK2. The hypothesis, formulated on the basis of preliminary data, is that the charge-triad motif on PED/PEA-15 maintains the essential flexibility of the DED, facilitating necessary conformational changes upon interacting with proteins involved in various biological pathways. The rationale for the proposed research is that, once the interaction between PED/PEA-15 and ERK is fully characterized, novel approaches to manipulate the interaction can likely be developed to curb tumor cell proliferation and invasion, enhancing the efficacy of anticancer drugs. The two specific aims of the project are: 1) Characterize the structure and conformation of PED/PEA-15 upon binding to ERK2; and 2) Illustrate roles of charge triad on PED/PEA-15 DED in recognition of ERK2. Under the first aim, the conformation of PED/PEA-15 in the complex with ERK2 will be solved using novel computational protocols incorporating experimental chemical shift and residue dipolar coupling (RDC) data. The binding interface between PED/PEA-15 and ERK2 will also be characterized using novel NMR techniques, including the newly developed RDC-based technique to monitor subtle conformational changes. Under the second aim, structural and dynamic studies of the ERK2-binding deficient D74A mutant of PED/PEA-15 and the homologous ERK2-binding protein vanishin/PEA-15b will be performed to delineate the roles of the charge triad on the binding capability. Both the mutant and vanishin/PEA-15b have disrupted charge-triad, and putatively lose the protein flexibility to some extent. Vanishin/PEA-15b structure, with R-CxDL triad, is expected more close to the ERK2-bound conformation, while the D74A mutant, with D-RxAL triad, is locked at the free-form conformation, and unable to bind ERK2. This research is innovative, because structural feature of charge triad is now linked to the function of maintaining the flexibility of the protein, instead of directly involved in the binding interface with other proteins. Several novel techniques, including the one being developed in the applicant's lab, are also used in the research. The proposed research is significant, because it is expected to vertically advance and expand understanding of protein-protein interactions in ERK pathway, which ultimately has the potential to develop novel intervening strategies of the ERK activity in the prevention and treatment of common cancers.